Cafeteria Diet-Induced Obesity Worsens Experimental CKD.

Obesity is a significant risk factor for chronic kidney disease (CKD). This study aimed to evaluate the impact of obesity on the development of kidney fibrosis in a model of cafeteria diet rats undergoing 5/6th nephrectomy (SNx). Collagen 1, 3, and 4 expression, adipocyte size, macrophage number, and the expression of 30 adipokines were determined. Collagen 1 expression in kidney tissue was increased in Standard-SNx and Cafeteria-SNx (7.1 ± 0.6% and 8.9 ± 0.9 tissue area, respectively). Renal expression of collagen 3 and 4 was significantly increased (p < 0.05) in Cafeteria-SNx (8.6 ± 1.5 and 10.9 ± 1.9% tissue area, respectively) compared to Cafeteria (5.2 ± 0.5 and 6.3 ± 0.6% tissue area, respectively). Adipocyte size in eWAT was significantly increased by the cafeteria diet. In Cafeteria-SNx, we observed a significant increase in macrophage number in the kidney (p = 0.01) and a consistent tendency in eWAT. The adipokine level was higher in the Cafeteria groups. Interleukin 11, dipeptidyl peptidase 4, and serpin 1 were increased in Cafeteria-SNx. In the kidney, collagen 3 and 4 expressions and the number of macrophages were increased in Cafeteria-SNx, suggesting an exacerbation by preexisting obesity of CKD-induced renal inflammation and fibrosis. IL11, DPP4, and serpin 1 can act directly on fibrosis and participate in the observed worsening CKD.

Implications of Senescent Cell Burden and NRF2 Pathway in Uremic Calcification: A Translational Study.

Increased senescent cell burden and dysregulation of the nuclear factor erythroid 2-related factor 2 (NRF2) pathway have been associated with numerous age-related pathologies; however, their role in promoting vascular calcification (VC) in chronic kidney disease (CKD) has yet to be determined. We investigated whether senescence and NRF2 pathways may serve as drivers of uremia-induced VC using three complementary approaches: a novel model of induced VC in 5/6-nephrectomized rats supplemented with high phosphate and vitamin D; epigastric arteries from CKD patients with established medial calcification; and vascular smooth muscle cells (VSMCs) incubated with uremic serum. Expression of p16Ink4a and p21Cip1, as well as γ-H2A-positive cells, confirmed increased senescent cell burden at the site of calcium deposits in aortic sections in rats, and was similarly observed in calcified epigastric arteries from CKD patients through increased p16Ink4a expression. However, uremic serum-induced VSMC calcification was not accompanied by senescence. Expression of NRF2 and downstream genes, Nqo1 and Sod1, was associated with calcification in uremic rats, while no difference was observed between calcified and non-calcified EAs. Conversely, in vitro uremic serum-driven VC was associated with depleted NRF2 expression. Together, our data strengthen the importance of senescence and NRF2 pathways as potential therapeutic options to combat VC in CKD.

Addressing the quality challenge of a human biospecimen biobank through the creation of a quality management system.

BACKGROUND: The objective of the COMET (COllection of MEtabolic Tissues) biobank project is to create a high-quality collection of insulin-sensitive tissues (liver, muscle, adipose tissues, and epiploic artery) and blood sample derivatives (plasma, serum, DNA and RNA), collected from 270 grade 2-3 obese patients undergoing bariatric surgery. Relevant data on patient such as clinical/biological characteristics and sample handling are also collected. For this, our aim was to establish a Quality Management System (QMS) to meet the reliability and quality requirements necessary for its scientific exploitation. MATERIALS AND METHODS: The COMET QMS includes: (1) Quality Assurance to standardize all stages of the biobanking process, (2) Quality Controls on samples from the first patients included in order to validate the sample management process and ensure reproducible quality; and 3) "in process" Quality Controls to ensure the reliability of the storage procedures and the stability of the samples over time. RESULTS: For serum and plasma, several corrective actions, such as temperature handling and centrifugation conditions, were made to the protocol and led to improvement of the volume and quality of samples. Regarding DNA, all samples evaluated achieved a satisfactory level of purity and integrity and most of them yielded the required DNA quantity. All frozen tissue samples had RNAs of good purity. RNA quality was confirmed by RIN, achieving values in most cases over 7 and efficient amplification of housekeeping genes by RT-qPCR, with no significant differences among samples from the same tissue type. In the "in process" Quality Controls, DNA, RNA, and histological integrity of tissues showed no differences among samples after different preservation times. CONCLUSION: Quality Control results have made it possible to validate the entire biobank process and confirm the utility of implementing QMS to guarantee the quality of a biospecimen collection.

Vascular calcification in different arterial beds in ex vivo ring culture and in vivo rat model.

Vascular calcification is a risk factor for cardiovascular and kidney diseases. Medial calcification may differently affect the arterial tree depending on vessel location and smooth muscle injury. The aim was to map the anatomical distribution of vascular calcifications on different arteries and artery locations, in cultured artery rings (ex vivo) and in a rat model of elastocalcinosis (in vivo). Vascular calcification was assessed histologically (von Kossa staining of the media) and by calcium content measurement. Arteries of different sizes were harvested from untreated rats for ring culture and from the vitamin D3-nicotine (VDN) rat model for direct observation. When cultured in pro-calcifying conditions, thoracic aorta exhibited similar calcification from the arch to the diaphragm. Calcification increased in abdominal aorta along with the reduction in cross sectional area. Carotid and renal arteries exhibited similar ex vivo calcification. In VDN rats, calcification was greater in carotid artery than in aorta, and was accompanied by fibrosis and apoptosis. Ex vivo, calcification was increased by the induction of lesions on arteries. Along the vascular tree, calcification of the arterial wall increases with the narrowing of vessels in ex vivo ring culture and in vivo. The observed differences represent local susceptibility of the vessels to the calcifying processes.

Reduced production of isoprostanes by peri-pancreatic adipose tissue from Zucker fa/fa rats as a new mechanism for ß-cell compensation in insulin resistance and obesity.

During early stages of type 2 diabetes, named prediabetes, pancreatic ß-cells compensate for insulin resistance through increased insulin secretion in order to maintain normoglycemia. Obesity leads to the development of ectopic fat deposits, among which peri-pancreatic white adipose tissue (pWAT) can communicate with ß-cells through soluble mediators. Thus we investigated whether pWAT produced oxygenated lipids, namely isoprostanes and neuroprostanes and whether they can influence ß-cell function in obesity. In the Zucker fa/fa rat model, pWAT and epididymal white adipose tissue (eWAT) displayed different inflammatory profiles. In obese rats, pWAT, but not eWAT, released less amounts of 5-F2t-isoprostanes, 15-F2t-isoprostanes, 4-F4t-neuroprostanes and 10-F4t-neuroprostane compared to lean animals. These differences could be explained by a greater induction of antioxidant defenses enzymes such as SOD-1, SOD-2, and catalase in pWAT of obese animals compared to eWAT. In addition, sPLA2 IIA, involved in the release of isoprostanoids from cellular membranes, was decreased in pWAT of obese animals, but not in eWAT, and may also account for the reduced release of oxidized lipids by this tissue. At a functional level, 15-F2t-isoprostane epimers, but not 5-F2t-isoprostanes, were able to decrease glucose-induced insulin secretion in pancreatic islets from Wistar rats. This effect appeared to be mediated through activation of the thromboxane A2 receptor and reduction of cAMP signaling in pancreatic islets. In conclusion, through the removal of an inhibitory tone exerted by isoprostanes, we have shown, for the first time, a new mechanism allowing ß-cells to compensate for insulin resistance in obesity that is linked to a biocommunication between adipose tissue and ß-cells.

Renal insufficiency and chronic kidney disease - Promotor or consequence of pathological post-translational modifications.

More than 840 million people, representing almost 10% of world population, were estimated to have chronic kidney disease (CKD) in 2017. In CKD, many systemic changes relative to oxidative stress, inflammation, energy balance or neuroendocrine signalling are observed and can be linked to dysfunctional proteins, including protein post-translational modifications (PTMs). Recent technical advances enabled the detection of PTMs and allowed understanding their participation in CKD pathophysiology and kidney damage. In this review article, the interconnections between CKD and PTMs, both as causes and consequences, are described. PTMs, particularly non-enzymatic PTMs, are frequently observed in CKD, as they are the direct consequence of systemic changes following the decline in kidney function. Other PTMs, mainly enzymatic ones, are critical for proper kidney physiology. Still, both types of PTMs have been shown to induce damage not only in kidney but also in other organs (brain, cardiovascular system). Therapeutic approaches focusing on metabolic changes responsible for PTMs alteration have shown interesting results. Targeting specific PTMs responsible for kidney damage is also being considered, which could lead to the development of innovative treatments.

Optimization of RNA extraction methods from human metabolic tissue samples of the COMET biobank.

Constitution of biobank of human tissues requires careful handling and storage of biological material, to guarantee the quality of samples. Tissue preparation is also critical for further applications such as transcriptomic profiling. In this study, our aim was to evaluate the impact of different disruption techniques (FastPrep-24 instrument, GentleMACS dissociator, and syringe/needle) and homogenizing buffers (RLT versus QIAzol) on RNA purity and quality of metabolic tissues (adipose tissues, liver and skeletal muscle) present in the COMET Biobank. For all homogenization methods used and tissue types, the A260/280 ratios reached values ≥ 1.8, which are in the range of what is found in human tissues and cell lines, while the A260/230 ratios were however ≤ 1.8, with the lowest value obtained with GentleMACS Dissociator. In addition, GentleMACS Dissociator combined with QIAzol reagent gave the highest RIN value and 28S/18S ratio for all tissues tested, except for muscle. Performing RT-qPCR, Ct values for different housekeeping genes can be influenced by extraction methods and RNA quality of samples. In conclusion, we have demonstrated that different disruption techniques and homogenizing buffers impact the purity and some quality markers of RNA, and can also impact quantification of mRNAs by RT-qPCR in human metabolic tissues.

Peripancreatic Adipose Tissue Remodeling and Inflammation during High Fat Intake of Palm Oils or Lard in Rats.

Excessive fat consumption leads to the development of ectopic adipose tissues, affecting the organs they surround. Peripancreatic adipose tissue is implicated in glucose homeostasis regulation and can be impaired in obesity. High palm oil consumption's effects on health are still debated. We hypothesised that crude and refined palm oil high-fat feeding may have contrasting effects on peripancreatic adipocyte hypertrophy, inflammation and lipid oxidation compound production in obese rats. In Wistar rats, morphological changes, inflammation and isoprostanoid production following oxidative stress were assessed in peripancreatic adipose tissue after 12 weeks of diets enriched in crude or refined palm oil or lard (56% energy from fat in each case) versus a standard chow diet (11% energy from fat). Epididymal white and periaortic brown adipose tissues were also included in the study. A refined palm oil diet disturbed glucose homeostasis and promoted lipid deposition in periaortic locations, as well as adipocyte hypertrophy, macrophage infiltration and isoprostanoid (5-F2c-isoprostane and 7(RS)-ST-Δ8-11-dihomo-isofuran) production in peripancreatic adipose tissue. Crude palm oil induced a lower impact on adipose deposits than its refined form and lard. Our results show that the antioxidant composition of crude palm oil may have a protective effect on ectopic adipose tissues under the condition of excessive fat intake.

The CKD plasma lipidome varies with disease severity and outcome.

BACKGROUND: Various alterations in lipid metabolism have been observed in patients with chronic kidney disease (CKD). OBJECTIVES: To determine the levels of lipid species in plasma from CKD and hemodialysis (HD) patients and test their association with CKD severity and patient outcome. METHODS: Seventy-seven patients with CKD stage 2 to HD were grouped into classes of CKD severity at baseline and followed-up for 3.5 years for the occurrence of transition to HD or death (combined outcome). Plasma levels of phosphatidylcholines (PCs), lysophosphatidylcholines (LPCs), sphingomyelins (SMs), and fatty acids were analyzed by flow-injection analysis coupled to tandem mass spectrometry or gas chromatography coupled with mass spectrometry. Kruskal Wallis rank tests and Cox regressions were used to analyze the association of lipids with CKD severity and the risk of combined outcome, respectively. RESULTS: The plasma level of PCs, LPCs, and SMs was decreased in HD patients compared with nondialyzed CKD patients (all P < .05), whereas esterified and/or nonesterified fatty acids level did not change. Thirty-four lipids displayed significantly lower abundance in plasma of HD patients, whereas elaidic acid (C18:1ω9t) level was increased (P < .001). The total amount of LPCs and individual LPCs were associated with better outcome (P < .05). In particular, LPC 18:2 and LPC 20:3 were statistically associated with outcome in adjusted models (P < .05). DISCUSSION: In HD patients, a reduction in plasma lipids is observed. Some of the alterations, namely reduced LPCs, were associated with the risk of adverse outcome. These changes could be related to metabolic dysfunctions.